Therapy for human cancers using cisplatin and other drugs or genes encapsulated into liposomes

ABSTRACT

A method for encapsulating cisplatin and other positively-charged drugs into liposomes having a different lipid composition between their inner and outer membrane bilayers is disclosed. The liposomes are able to reach primary tumors and their metastases after intravenous injection to animals and humans. The encapsulated cisplatin has a high therapeutic efficacy in eradicating a variety of solid human tumors including but not limited to breast carcinoma and prostate carcinoma. Combination of the encapsulated cisplatin with encapsulated doxorubicin or with other antineoplastic drugs are claimed to be of therapeutic value. Also of therapeutic value in cancer eradication are claimed to be combinations of encapsulated cisplatin with a number of anticancer genes including but not limited to p53, IL-2, IL-12, angiostatin, and oncostatin encapsulated into liposomes as well as combinations of encapsulated cisplatin with HSV-tk plus encapsulated ganciclovir.

FIELD OF THE INVENTION

The present invention relates to liposome encapsulated drugs anddelivery systems, specifically liposome encapsulated cisplatin. Thedrugs are useful to kill cancer cells in a variety of human malignanciesafter intravenous injection.

BACKGROUND OF THE INVENTION

Throughout this application various publications, patents and publishedpatent specifications are referenced by author and date or by anidentifying patent number. Full bibliographic citations for thepublications are provided within this disclosure or immediatelypreceding the claims. The disclosures of these publications, patents andpublished patent specifications are hereby incorporated by referenceinto the present disclosure to more fully describe the state of the artto which this invention pertains.

Cis-diamminedichloroplatinum(II), cis-[Pt(NH₃)₂Cl₂]²⁺, abbreviatedcisplatin or cis-DDP, is one of the most widely used antineoplasticdrugs for the treatment of testicular, ovarian carcinomas and againstcarcinomas of the head and neck. More than 90% of testicular cancers arecured by cisplatin. The most severe side effects are nephrotoxicity,bone marrow toxicity and gastrointestinal irritation (Oliver and Mead,1993). Bilateral optic neuropathy was observed in a patient affected byovarian carcinoma treated with 160 mg/m² cisplatin and 640 mg/m²carboplatin (Caraceni et al., 1997). Oral hexamethylmelamine treatmentin a group of 61 patients with epithelial ovarian carcinoma with cis- orcarboplatin resistance (relapse within 6 months after the end of thattherapy) showed a 14% objective response rate (Vergote et al., 1992).

Cationic cholesterol derivatives have been used to deliver therapeuticagents. For example, they have been mixed with phosphatidylethanolamineand sonicated to form small unilamellar vesicles which can complex withDNA and mediate the entry into the cytosol from the endosomecompartment. One of the liposome formulations, DC-Chol liposomes, hasbeen used in a gene therapy clinical trial against melanoma. Humanimmunodeficiency virus-1 transactivating protein gene was codeliveredwith a reporter gene under the control of HIV-1 long terminal repeat.Human tumor cells selected for cisplatin resistance or isolated frompatients who had failed cisplatin therapy were highly transfectable withcationic liposomes. These results suggested a serial therapy protocolwith cisplatin and gene therapy for malignancy (Farhood et al., 1994).

Various platinum complexes prepared from 2-amino-methylpyrrolidinederivatives as carrier ligands were tested for their antitumor activityagainst Colon 26 carcinoma and P388 leukemia using subcutaneous and/orintraperitoneal injections in mice. 2-aminomethylpyrrolidine proved tobe the most effective carrier ligand in its amine derivatives (Morikawaet al., 1990).

An optimum procedure was established by orthogonal test for preparingcisplatin albumin microspheres (Cis-DDP-AMS) with emulsion-heatingstabilization method (mean size was 148 microns). The distribution andelimination half times of platinum were prolonged 3.36 times and 1.23times after hepatic arterial chemoembolization with Cis-DDP-AMS versusCis-DDP, respectively (Zhang et al., 1995).

The search for platinum (II)-based compounds with improved therapeuticproperties was prompted to design and synthesize a new family ofwater-soluble, third generation cis-diaminedichloroplatinum (II)complexes linked to uracil and uridine. However, none of the synthesizedcompounds showed any significant cytotoxic activity against three celllines that were treated (Kim et al., 1998).

The recently developed bioreductive agent4-[3-(2-nitroimidazolyl)-propylamino]-7-chloroquinoline hydrochloride(NLCQ-1) was found to potentiate the antitumor effect of thechemotherapeutic agents melphalan (L-PAM), cisplatin (cisDDP) andcyclophosphamide (CPM) without concurrent enhancement in bone marrowtoxicity. Potentiation was strictly schedule dependent and the optimumeffect (1.5 to 2 logs killing beyond additivity) was observed whenNLCQ-1 was given 45-min before cisDDP. These results support theclassification of NLCQ-1, based on clinical studies, as achemosensitizer (Papadopoulou et al., 1998).

A combination of paciltaxel with cisplatin as second-line treatment inpatients with non-small cell lung cancer (NSCLC) who had previouslyundergone first-line therapy with cisplatin achieved partial response(40%) in 14 patients (Stathopoulos et al., 1999).

Abra et al. (U.S. Pat. No. 5,945,122, issued Aug. 31, 1999) describes aliposome composition containing entrapped non-charged cisplatin inmostly neutral lipids. However, the process of Abra et al. uses neutrallipids compared with the anionic lipid DPPG disclosed in the presentpatent for cisplatin entrapment.

Thus, while the prior reports indicate that liposome mediated deliveryof cisplatin and other therapeutic drugs is possible, therapeuticefficiency has been limited by the low aqueous solubility and lowstability of cisplatin. Therapeutic efficacy also is limited by the hightoxicity of the drug. Thus, a need exists to reduce the difficultiesinvolved in processing of cisplatin containing drugs and high toxicityof cisplatin when used therapeutically. This invention satisfies thisneed and provides related advantages as well.

DISCLOSURE OF THE INVENTION

In one aspect, this invention provides a method for encapsulatingcisplatin and other positively-charged drugs into liposomes having adifferent lipid composition between their inner and outer membranebilayers and able to reach primary tumors and their metastases afterintravenous injection to animals and humans. In one aspect, the methodincludes complex formation between cisplatin with DPPG (dipalmitoylphosphatidyl glycerol) or other lipid molecules to convert cisplatin toits aqua form by hydrolysis which is positively-charged and is theactive form of cisplatin endowed with the antineoplastic activity. Atthis stage membrane fusion peptides and other molecules with fusogenicproperties may be added to improve entrance across the cell membrane ofthe complex. The aqua cisplatin-DPPG micelles are converted intoliposomes by mixing with vesicle forming lipids such as pre-madeliposomes or lipids followed by dialysis and extrusion throughmembranes, entrapping and encapsulating cisplatin to a very high yield.Doxorubicin or other positively-charged compounds can be substituted forcisplatin in these formulations. The encapsulated cisplatin has a hightherapeutic efficacy in eradicating a variety of solid human tumorsincluding but not limited to breast carcinoma and prostate carcinoma.Combination of the encapsulated cisplatin with encapsulated doxorubicinor with other antineoplastic drugs are claimed to be of therapeuticvalue. Also of therapeutic value in cancer eradication are claimed to becombinations of encapsulated cisplatin with a number of anticancer genesincluding but not limited to p53, IL-2, IL-12, angiostatin, andoncostatin encapsulated into liposomes as well as combinations ofencapsulated cisplatin with HSV-tk plus encapsulated ganciclovir.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts cisplatin encapsulation.

FIG. 2 shows MCF-7 tumor regression in SCID mice after 3-4 injections ofencapsulated cisplatin.

FIG. 3 shows histology of tumors in SCID mice with or without treatmentwith cisplatin.

FIG. 3A shows untreated MCF-7 tumors grown in SCID mice. 40×magnification. Notice the homogeneous pattern of structurescharacteristic of tumor tissue.

FIG. 3B shows cisplatin-treated mice (4 injections). Cells areapoptotic, there are groups of cells into structures and nuclei stainbigger and darker, characteristic of apoptotic cells.

FIG. 3C shows tumors from untreated animals showing invasion to muscle.20×.

FIG. 3D shows cisplatin-treated mice. Invasion is not evident. 20×magnification.

FIG. 4 shows macroscopic (visual) difference in tumor size between ananimal treated with encapsulated cisplatin (A) and an untreated animal(B)).

MODES FOR CARRYING OUT THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA. These methods aredescribed in the following publications. See, e.g., Sambrook, et al.MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.); “PCR: A PRACTICALAPPROACH” (M. MacPherson, et al., IRL Press at Oxford University Press(1991)); PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames andG. R. Taylor eds. (1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow andLane, eds. (1988)); and ANIMAL CELL CULTURE (R. I. Freshney, ed.(1987)).

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example, single-,double-stranded and triple helical molecules, a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A nucleic acid molecule may also comprise modified nucleicacid molecules.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular polypeptide or proteinafter being transcribed and translated.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label or a pharmaceutically acceptable carrier) or active, such as anadjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'SPHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

A “subject,” “individual” or “patient” is used interchangeably herein,which refers to a vertebrate, preferably a mammal., more preferably ahuman. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative.” Forexample, where the purpose of the experiment is to determine acorrelation of an altered expression level of a gene with a particulartype of cancer, it is generally preferable to use a positive control (asubject or a sample from a subject, carrying such alteration andexhibiting syndromes characteristic of that disease), and a negativecontrol (a subject or a sample from a subject lacking the alteredexpression and clinical syndrome of that disease).

A “gene delivery vehicle” is defined as any molecule that can carryinserted polynucleotides into a host cell. Examples of gene deliveryvehicles are liposomes, cationic liposomes, viruses, such asbaculovirus, adenovirus, adeno-associated virus, and retrovirus,bacteriophage, cosmid, plasmid, fungal vectors and other recombinationvehicles typically used in the art which have been described forexpression in a variety of eukaryotic and prokaryotic hosts, and may beused for gene therapy as well as for simple protein expression.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors and the like. In aspects where gene transfer is mediated by aretroviral vector, a vector construct refers to the polynucleotidecomprising the retroviral genome or part thereof, and the insertedpolynucleotide. As used herein, “retroviral mediated gene transfer” or“retroviral transduction” carries the same meaning and refers to theprocess by which a gene or nucleic acid sequences are stably transferredinto the host cell by virtue of the virus entering the cell andintegrating its genome into the host cell genome. The virus can enterthe host cell via its normal mechanism of infection or be modified suchthat it binds to a different host cell surface receptor or ligand toenter the cell. As used herein, retroviral vector refers to a viralparticle capable of introducing exogenous nucleic acid into a cellthrough a viral or viral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form, which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a polynucleotide to be inserted. Adenoviruses (Ads)are a relatively well characterized, homogenous group of viruses,including over 50 serotypes. (see, e.g., WO 95/27071). Ads are easy togrow and do not require integration into the host cell genome.Recombinant Ad-derived vectors, particularly those that reduce thepotential for recombination and generation of wild-type virus, have alsobeen constructed. (see, WO 95/00655; WO 95/11984). Wild-type AAV hashigh infectivity and specificity integrating into the host cells genome.(Hermonat and Muzyczka (1984) PNAS USA 81:6466-6470; Lebkowski, et al.(1988) Mol. Cell. Biol. 8:3988-3996).

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include several non-viral vectors, includingDNA/liposome complexes, and targeted viral protein DNA complexes.Liposomes that also comprise a targeting antibody or fragment thereofcan be used in the methods of this invention. To enhance delivery to acell, the nucleic acid or proteins of this invention can be conjugatedto antibodies or binding fragments thereof which bind cell surfaceantigens, e.g., TCR, CD3 or CD4.

Polynucleotides are inserted into vector genomes using methods wellknown in the art. For example, insert and vector DNA can be contacted,under suitable conditions, with a restriction enzyme to createcomplementary ends on each molecule that can pair with each other and bejoined together with a ligase. Alternatively, synthetic nucleic acidlinkers can be ligated to the termini of restricted polynucleotide.These synthetic linkers contain nucleic acid sequences that correspondto a particular restriction site in the vector DNA. Additionally, anoligonucleotide containing a termination codon and an appropriaterestriction site can be ligated for insertion into a vector containing,for example, some or all of the following: a selectable marker gene,such as the neomycin gene for selection of stable or transienttransfectants in mammalian cells; enhancer/promoter sequences from theimmediate early gene of human CMV for high levels of transcription;transcription termination and RNA processing signals from SV40 for mRNAstability; SV40 polyoma origins of replication and ColEI for properepisomal replication; versatile multiple cloning sites; stabilizingelements 3′ to the inserted polynucleotide, and T7 and SP6 RNA promotersfor in vitro transcription of sense and antisense RNA. Other means arewell known and available in the art.

“Host cell” is intended to include any individual cell or cell culturewhich can be or have been recipients for vectors or the incorporation ofexogenous polynucleotides, polypeptides and/or proteins. It also isintended to include progeny of a single cell, and the progeny may notnecessarily be completely identical (in morphology or in genomic ortotal DNA complement) to the original parent cell due to natural.,accidental., or deliberate mutation. The cells may be prokaryotic oreukaryotic, and include but are not limited to bacterial cells, yeastcells, plant cells, insect cells, animal cells, and mammalian cells,e.g., murine, rat, simian or human.

As used herein, the terms “neoplastic cells,” “neoplasia,” “tumor,”“tumor cells,” “cancer” and “cancer cells,” (used interchangeably) referto cells which exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation (i.e., de-regulated cell division).Neoplastic cells can be malignant or benign.

“Suppressing” tumor growth indicates a growth state that is curtailedwhen compared to control cells. Tumor cell growth can be assessed by anymeans known in the art, including, but not limited to, measuring tumorsize, determining whether tumor cells are proliferating using a³H-thymidine incorporation assay, or counting tumor cells. “Suppressing”tumor cell growth means any or all of the following states: slowing,delaying, and stopping tumor growth, as well as tumor shrinkage.

Embodiments of the Invention

Micelles, Liposomes and Processes for Obtaining Them

Claimed herein is a new method for entrapping cisplatin into lipidswhich enhances the content of cisplatin per volume unit, reduces itstoxicity, is able to target primary tumors and their metastases afterintravenous injection, and shows shrinkage of tumors and completetherapy of SCID mice bearing human tumors.

Cisplatin is a heavy metal complex containing two chloride atoms and twoamino groups in the cis position attached to one atom of the transitoryheavy metal platinum in its divalent form. It is a bifunctionalalkylating agent as well as DNA intercalator inhibiting DNA synthesis.In one form, cisplatin is a yellow powder of a molecular weight of 300.1and of limited solubility of 1 mg/ml in water. It is widely used for thetreatment of cancer patients, especially those of testicular, lymphomas,endometrial, bladder, ovarian, head and neck squamous cell carcinomas,breast carcinomas, and many other malignancies, often in combinationwith adriamycin, vinblastin, bleomycin, prednisone, vincristine, taxol,and others antineoplastic drugs as well as radiation therapy. We claimreduction in total cisplatin volume required for patient treatmentbecause of an increase in its solubility in its lipid entrapment form

The volume used for intravenous injection is usually large (about 180 mlper adult patient or about 20-120 mg/m) administered as a 24-hourinfusion. It is cleared from the plasma in a rapid phase of 25-80 minfollowed by a slower secondary phase of 58-73 h; it is bound by plasmaproteins and excreted by the kidneys (explaining the severe kidneytoxicity in treated patients). Dose related nephrotoxicity can bepartially overcome with vigorous hydration, mannitol, furosemide andother drugs. Other toxicities incurred by cisplatin include ototoxicity,nausea and vomiting, anemia, and mild myelosuppression (The Merck Manualof Diagnosis and Therapy). The present invention overcomes thelimitations of prior art processes and compositions.

Thus, in one aspect, this invention provides methods for producingcisplatin micelles, by combining cisplatin and a phosphatidyl glycerollipid derivative (PGL derivative) in a range of 1:1 to 1:2.1 to form acisplatin mixture. In alternative embodiments, the range of cisplatin toPGL derivative is in the ranges 1:1.2; or 1:1.4; or 1:1.5; or 1:1.6; or1:1.8 or 1:1.9 or 1:2.0 or 1:2.1. The mixture is then combined with aneffective amount of at least a 20% organic solvent such as an ethanolsolution to form cisplatin micelles.

As used herein, the term “phosphatidyl glycerol lipid derivative (PGLderivative)” is any lipid derivative having the ability to form micellesand have a net negatively charged head group. This includes but is notlimited to dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoylphosphatidyl glycerol, and dicapryl phosphatidyl glycerol. In oneaspect, phosphatidyl derivatives with a carbon chain of 10 to 28 carbonsand having unsaturated side aliphatic side chain are within the scope ofthis invention. The complexation of cisplatin with negatively-chargedphosphatidyl glycerol lipids having variations in the molar ratio givingthe particles a net positive (1:1) neutral (1:2) or slightely negative(1:2.1 ) charge will allow targeting of different tissues in the bodyafter administration. However, complexing of cisplatin with negativelycharged PGL has been shown to enhance the solubility of cisplatin, thusreducing the volume of the drug required for effective antineoplastictherapy. In addition, the complexation of cisplatin and negativelycharged PGL proceeds to very high encapsulation efficiency minimizingdrug loss during the manufacturing process. These complexes are stable,do not form precipitates and retain therapeutic efficacy after storageat 4° C. for at least 4 months.

As used herein, the term “cisplatin” included analogs. Examples includecarboplatin, ormaplatin, oxaplatin, 2-aminomethylpyrrolidine(1,1-cyclobutane dicarboxylato)platinum, lobaplatin,1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N′)platinum,zeniplatin, enloplatin, 254-S nedaplatin and JM-216(bis-acetato-amine-dichloro-cyclohexylamine-platinum(IV)).

It is to be understood, although not always explicitly stated, thatother positively charged drugs, including but not limited to theantineoplastic drug doxorubicin can be substituted for cisplatin.Alternatively, other types of drugs that are neutral can be used upontheir conversion into positively charged drugs by derivation withpositively charged groups. Modification of a neutral ornegatively-charged anticancer or other type of drug to apositively-charged molecule can be accomplished by a number of methodswell established in organic synthesis. This can be achieved for exampleby replacing a hydroxyl group in the drug with an amino group or by atrimethylamino group thus introducing a positive charge to the compound.Replacement of a ring hydroxyl group with an amino group is discussed inU.S. Pat. No. 5,837,868, Wang, et al. issued Nov. 17, 1998.

The above method does not require that the steps be performed in theorder indicated above. For example, the method can be practiced bycombining cisplatin with an effective amount of at least a 20% organicsolvent solution to form a solution. The solution is combined with aphosphatidyl glycerol lipid (PGL) derivative in a range of 1:1 to 1:2.1to form a cisplatin micelle. As above, the range of cisplatin to PGLderivative is in the ranges 1:1.2; or 1:1.4; or 1:1.5; or 1:1.6; or1:1.8 or 1:1.9 or 1:2.0 or 1:2.1.

Any organic solvent or formulation of ethanol, or any other alcohol thatdoes not form a two phase system, or other organic solvent (i.e.,choroform), that is miscible in 20% alcohol, is useful in the methodsdescribed herein. For example, the alcohol solution can be any of atleast 30%, 35%, 40%, 45% up to 90%, including any increment in between.Preferably, the alcohol solution is 30% ethanol for DPPG, and for otherlipids the optimal percentage may be different.

In one embodiment, partial replacement of DPPG molecules by peptideswith a net negative charge gives to cisplatin complexes having fusogenicproperties able to cross the cell membrane of the target. Fusogenicpeptides may also be covalently attached at the free and of PEG fortheir better exposure. Addition of a small amount of cationic lipidsreplacing positive charges of aqua cisplatin at the final cisplatin/DPPGcomplex also endows the complex with fusogenic properties; thepercentage of positive charges to be substituted by cationic lipids(e.g., DDAB, dimethyldioctadecyl ammonium bromide; DMRIE:N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammoniumbromide; DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane; DOGS:Dioctadecylamidoglycylspermine; DOTAP:N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DPTAP:1,2-dipalmitoyl-3-trimethylammonium propane; DSTAP:1,2-disteroyl-3-trimethylammonium propane) is small because of thetoxicity of cationic lipids. Some formulations contain an amount of thefusogenic amphiphilic lipid DOPE in the micelles.

In a further aspect, the cisplatin micelles are encapsulated intovesicle forming lipids, for example, for use in drug delivery.

The lipid encapsulated cisplatin has a high therapeutic efficacy ineradicating a variety of solid human tumors including but not limited tobreast carcinoma, prostate carcinoma, glioblastoma multiform, non-smalllung cell carcinoma, pancreatic carcinoma, head and neck squamous cellcarcinoma and T-cell lymphomas. Accordingly in another aspect, theinvention provides a method for the treatment of a variety of humanmalignancies using an encapsulated cisplatin or alternatively, otherpositively-charged antineoplastic drugs into liposomes having adifferent lipid composition between their inner and outer membranebilayers. The liposome encapsulated drugs are able to reach primarytumors and their metastases after intravenous injection to animals andhumans.

A combination of the encapsulated cisplatin with doxorubicin or withother antineoplastic drugs have a higher therapeutic efficacy thancisplatin alone. Also of higher therapeutic efficacy in cancereradication are combinations of encapsulated cisplatin with a number ofanticancer genes including but not limited to p53, IL-2, IL-12,angiostatin, and oncostatin encapsulated into similar type of liposomesas well as combinations of encapsulated cisplatin with HSV-tk plusencapsulated ganciclovir.

In a further aspect, the invention provides a combination of theencapsulated cisplatin with the following genes:

(i) A wild-type (wt) p53 cDNA expression vector under control of theCMV, beta-actin, or other promoters, and human origins of replicationable to sustain long term expression of the p53 gene; viral origins ofreplication which require viral replication initiator proteins such as Tantigen for their activation are nor suitable for the transfer of thep53 gene because p53 protein interacts strongly with T antigen.

(ii) A PAX5 cDNA expression vector, the only suppressor of the p53 geneknown (both of the wt and mutant p53 genes) interacting with a short (10nucleotide) regulatory region within intron 1 of the p53 gene. A majordrawback in p53 gene therapy is the inactivation of the wt p53 proteinby the endogenous mutated forms of p53 which are overexpressed in tumorsand which are able to tetramerize with wt p53 protein; the endogenousp53 genes will be suppressed by expression of Pax5, a potenttranscriptional repressor of the p53 gene. The wt p53 cDNA vector lacksintron 1 and by consequence the suppressive PAX5 binding region. It isimportant to suppress the endogenous mutant p53 gene expression andeliminate mutant p53 from the cancer cells to potentiate induction ofapoptosis and tumor suppression.

(iii) The herpes simplex virus thymidine kinase (HSV-tk) gene. Theherpes simplex virus thymidine kinase (HSV-tk) suicide gene will be alsoincluded in combinations of p53 and Pax5 genes causing interruption inDNA synthesis after ganciclovir (GCV) treatment of the animal model andhuman patient; this is expected to increase the strand breaks in thecancer cells and to potentiate the tumor suppressor functions of p53known to bind to strand breaks and to damaged DNA sites. In a furtherembodiment, ganciclovir is combined and encapsulated into liposomes.

Gene therapy is a new era of biomedical research aimed at introducingtherapeutic genes into somatic cells of patients (reviewed by Boulikas,1998a; Martin and Boulikas, 1998). Two major obstacles prohibitsuccessful application of somatic gene transfer: (1) the smallpercentage of transduced cells and (2) the loss of the transcriptionsignal of the therapeutic gene after about 3-7 days from injection invivo.

The first problem arises (i) from inability of delivery vehiclescarrying the gene to reach the target cell surface (the vast majority ofliposome-DNA complexes are eliminated from blood circulation rapidly);(ii) from difficulty to penetrate the cell membrane and (iii) to releasethe DNA from endosomes after internalization by cells; (iv) frominefficient import into nuclei. Others have used stealth liposomes(Martin and Boulikas, 1998a), which persist in circulation for days andconcentrate in tumors. However, classical stealth liposomes are nottaken up by cancer cells. Disclosed herein are strategies that aredesigned to enhance liposome internalization (fusogenic peptides).

The second problem results from the loss of the plasmids in the nucleusby nuclease degradation and failure to replicate autonomously leading totheir dilution during cell proliferation among progeny cells or byinactivation of the foreign DNA after integration into the chromosomesof the host cell. However, the use of human sequences able to sustainextrachromosomal replication of plasmids for prolonged periods (see U.S.Patent on “Cloning method for trapping human origins of replication” byTeni Boulikas U.S. Pat. No. 5,894,060) will overcome this limitation.

Also claimed herein are tumor regression and reduction in tumor massvolume of breast, prostate and other cancers in animal models and inhumans after delivery of encapsulated cisplatin (termed Lipoplatin™) orencapsulated doxorubicin, and combinations of these drugs with genesincluding but not limited to the p53, PAX5, and HSV-tk/encapsulatedganciclovir, IL-2, IL-12, GM-CSF, angiostatin, IL-4, IL-7, IFN-gamma,TNF-alpha, RB, BRCA1, E1A, cytosine deaminiase in combination withencapsulated 5-fluorocytosine, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F,IGF-I VEGF, TGF-beta genes and the like.

Accordingly, this invention also provides a method for deliveringcisplatin or other therapeutic agent to a cell comprising contacting thecell with the encapsulated drugs or other intermediate, obtainable bythe methods of this invention. Also provided by this invention is amethod for inhibiting the growth of a tumor in a subject, comprisingadministering to the subject an effective amount of the encapsulateddrugs obtainable by the methods of this invention. The methods can bepracticed in vitro, ex vivo or in vivo.

Thus, this invention also provides combination therapy usingencapsulated drugs and polynucleotides. As used herein, apolynucleotides includes but is not limited to genes encoding proteinsand polypeptides as well as sequences encoding ribozymes and antisense.The combination therapy is more effective in eradicating cancer thaneither treatment alone because the two mechanisms are different and canachieve a synergism. For example, the property of p53 protein to bind todamaged DNA regions and free ends of DNA is known and also to triggerthe mechanism of apoptosis in severely-damaged cells (reviewed byBoulikas, 1998a). Free ends of DNA in cancer cells are expected to beproduced after damage by cisplatin enhancing the induction of anapoptotic pathway in these cells by the expression of the transferred wtp53 (many tumors have mutated p53 and might be unable to induceeffectively this pathway). The therapeutic polynucleotide also can beinserted into a gene transfer vector prior to incorporation into themicelle.

Transfer of the wild-type p53 gene has been successfully used toslow-down tumor cell proliferation in vivo and in cell culture innumerous studies. Intratumoral injection using adenoviral/p53 vectorshas been shown to be effective against lung tumors in recent clinicaltrials (Roth et al., 1996) and against prostate tumors on animal models(reviewed by Boulikas, 1998a). The intratumoral injection method,however, may not be applicable to metastases often associated with latestages of cancer. Systemic delivery of the p53 gene with encapsulatedcisplatin and targeting of tumors in any region of the body is aneffective treatment for cancer cure. We claim strategies forameliorating or partially overcoming the four main obstacles forsuccessful somatic gene transfer using liposomal delivery of the wt p53,pax5, HSV-tk, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-gamma, TNF-alpha, RB,BRCA1, E1A, cytosine deaminiase in combination with encapsulated5-fluorocytosine, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF,TGF-beta, angiostatin and other genes in combination with encapsulatedcisplatin and other drugs to a variety of human cancers in animal modelsand in patients to be tested in clinical trials. These include: (i)concentration and encapsulation of the drug and gene bullets intoliposomes reducing their toxicity; (ii) targeting of solid tumors andmetastases by coating of the surface of the complexes with polyethyleneglycol (PEG), hyaluronic acid or other polymers; (iii) enhancement inuptake of drugs and plasmids by cancer cells because of the fusogenicpeptides or small percentage of cationic lipids, and (iv) sustainedexpression of the genes using human origins of replication (ORIs) ableto sustain episomal replication or long term expression of therapeuticgenes and high levels of expression.

In a further embodiment, the polynucleotides or genes further compriseregulatory DNA sequences that sustain expression of the genes for monthsrather than days. This translates into fewer treatments and lesssuffering of the cancer patient. It can also exert a strong therapeuticeffect because of higher levels of expression of the anticancer gene;the same gene placed under control of weak regulatory DNA will beineffective.

Several experimental strategies for cancer treatment have been designedusing p53 gene delivery; our novelty consists in that the endogenousmutant p53 forms, which are overexpressed in over half of human prostatemalignancies especially those from advanced prostate cancer aresuppressed using the PAX5 expression vector. Mutated forms of p53 haveamino acid substitutions mainly in their DNA binding domain but arestill able to tetramerize with the wt p53 form; p53 acts as a tetramerand the presence of high levels of endogenous mutant p53 in humancancers cells interferes with the tumor suppressor functions of the wtp53 to be delivered.

PAX5 is an homeodomain protein which determines body structures duringdevelopment; PAX5 is expressed at early stages of mammalian developmentand in the adult during differentiation in hematopoietic stem cells; p53gene expression is eliminated by the PAX5 suppressor protein at earlystages of development allowing cells to multiply fast in the developingembryo. PAX5 is switched off at later stages throughout adulthoodallowing p53 to be expressed and exert its tumor suppressive functionsand to regulate apoptosis especially in the hematopoietic cell lineage.

A number of delivery systems are being used in somatic gene transfer,each associated with advantages and drawbacks. Recombinant adenovirusesdo not replicate efficiently; recombinant murine retroviruses integraterandomly and are inactivated by chromatin surroundings; recombinant AAVintegrates randomly and cannot achieve high titers for clinical utility.All have a maximum capacity of 3.5-7.5 kb of foreign DNA because ofpackaging limitations. Naked DNA is rapidly degraded (half-life 5minutes) after systemic delivery. Cationic liposomes are toxic do notsurvive in circulation beyond a heart beat and target mainly theendothelium of the lung, liver, and heart. So far, only “stealth”liposomes have been proven capable of concentrating in tumor sites (alsoin liver and spleen) and to survive for prolonged periods in bloodcirculation (e.g., one day compared with minutes for non-stealth neutralliposomes and a few seconds for cationic liposomes). However, stealthliposomes are not taken readily by tumor cells remaining in theextracellular space where they release their load over days after lysis(reviewed by Martin and Boulikas, 1998); however, the aspect of theinvention described below modifies stealth liposomes with fusogenicpeptides or by providing a partially cationic lipid composition or DOPEat their inner bilayer which would endow them to enter the tumor cellmembrane by causing disturbance of the lipid bilayer.

Having attained concentration and uptake of the drug and gene bullets insolid tumors in animals with stealth liposomes, the second step isefficacy of our drug and gene targeting approach. A human clinical trialat M. D. Anderson Cancer Center uses transfer of the wild-type p53 genein patients suffering with non-small cell lung cancer and shown to havep53 mutations in their tumors using local injection of an Ad5/CMV/p53recombinant adenovirus at the site of tumor in combination withcisplatin. The first results of this clinical trial are encouragingafter intratumor injection of p53 (Roth et al., 1996; reviewed byBoulikas, 1998a). However, local injection is not applicable tometastases often associated with advanced stages of malignancies; inparticular, prostate cancer gives metastases to bones by a mechanisminvolving stimulation in prostate tumor proliferation by insulin-likegrowth factor I (IGF-I) which is especially secreted by bone cells.Therefore, the delivery system proposed here, able to concentrate intothe tumor cell mass after systemic injection, is likely to treat notonly the primary tumor but also its metastases.

The proposed cisplatin liposomes will primarily target tumors because ofthe nature of our delivery system. The genes in the combination therapywill primarily target dividing cells because of the use of HSV-tk andganciclovir that incorporates into replicating DNA, and primarilyvascularizing tumors because of the use of stealth liposomes. Thus,liver and spleen cells that are also reached by stealth liposomes willnot be killed.

In a further embodiment, the liposome encapsulated drugs describedherein further comprise an effective amount of a fusogenic peptide.Fusogenic peptides belong to a class of helical amphipathic peptidescharacterized by a hydrophobicity gradient along the long helical axis.This hydrophobicity gradient causes the tilted insertion of the peptidesin membranes, thus destabilizing the lipid core and, thereby, enhancingmembrane fusion (Decout et al., 1999).

Hemagglutinin (HA) is a homotrimeric surface glycoprotein of theinfluenza virus. In infection, it induces membrane fusion between viraland endosomal membranes at low pH. Each monomer consists of thereceptor-binding HA1 domain and the membrane-interacting HA2 domain. TheNH2-terminal region of the HA2 domain (amino acids 1 to 127), theso-called “fusion peptide,” inserts into the target membrane and plays acrucial role in triggering fusion between the viral and endosomalmembranes. Based on substitution of eight amino acids in the region 5-14with cysteines and spin-labeling electron paramagnetic resonance it wasconcluded that the peptide forms an alpha-helix tilted approximately 25degrees from the horizontal plane of the membrane with a maximum depthof 15 angstroms (A) from the phosphate group (Macosko et al., 1997). Useof fusogenic peptides from influenza virus hemagglutinin HA-2 enhancedgreatly the efficiency of transferrin-polylysine-DNA complex uptake bycells; in this case the peptide was linked to polylysine and the complexwas delivered by the transferrin receptor-mediated endocytosis (reviewedby Boulikas, 1998a). This peptide had the sequence:GLFEAIAGFIENGWEGMIDGGGYC (SEQ ID NO:1) and was able to induce therelease of the fluorescent dye calcein from liposomes prepared with eggyolk phosphatidylcholine which was higher at acidic pH; this peptide wasalso able to increase up to 10-fold the anti-HIV potency of antisenseoligonucleotides, at a concentration of 0.1-1 mM, using CEM-SSlymphocytes in culture. This peptide changes conformation at theslightly more acidic environment of the endosome destabilizing andbreaking the endosomal membrane (reviewed by Boulikas, 1998a).

The presence of negatively charged lipids in the membrane is importantfor the manifestation of the fusogenic properties of some peptides butnot of others; whereas the fusogenic action of a peptide, representing aputative fusion domain of fertilin, a sperm surface protein involved insperm-egg fusion, was dependent upon the presence of negatively chargedlipids. However, that of the HIV2 peptide was not (Martin andRuysschaert, 1997).

For example, to analyze the two domains on the fusogenic peptides ofinfluenza virus hemagglutinin HA, HA-chimeras were designed in which thecytoplasmic tail and/or transmembrane domain of HA was replaced with thecorresponding domains of the fusogenic glycoprotein F of Sendai virus.Constructs of HA were made in which the cytoplasmic tail was replaced bypeptides of human neurofibromin type 1 (NF1) (residues 1441 to 1518) orc-Raf-1, (residues 51 to 131). The constructs were expressed in CV-1cells by using the vaccinia virus-T7 polymerase transient-expressionsystem. Membrane fusion between CV-1 cells and bound human erythrocytes(RBCs) mediated by parental or chimeric HA proteins showed that, afterthe pH was lowered, a flow of the aqueous fluorophore calcein frompreloaded RBCs into the cytoplasm of the protein-expressing CV-1 cellstook place. This indicated that membrane fusion involves both leafletsof the lipid bilayers and leads to formation of an aqueous fusion pore(Schroth-Diez et al., 1998).

A remarkable discovery was that the TAT protein of HIV is able to crosscell membranes (Green and Loewenstein, 1988) and that a 36-amino aciddomain of TAT, when chemically crosslinked to heterologous proteins,conferred the ability to transduce into cells. It is worth mentioningthat the 11-amino acid fusogenic peptide of TAT (YGRKKRRQRRR (SEQ IDNO:2)) is a nucleolar localization signal (see Boulikas, 1998b).

Another protein of HIV, the glycoprotein gp41, contains fusogenicpeptides. Linear peptides derived from the membrane proximal region ofthe gp41 ectodomain have potential applications as anti-HIV agents andinhibit infectivity by adopting a helical conformation (Judice et al.,1997). The 23 amino acid residues N-terminal peptide of HIV-1 gp41 hasthe capacity to destabilize negatively charged large unilamellarvesicles. In the absence of cations the main structure was apore-forming alpha-helix, whereas in the presence of Ca2⁺ theconformation switched to a fusogenic, predominantly extended beta-typestructure. The fusion activity of HIV(ala) (bearing the R22(Asubstitution) was reduced by 70% whereas fusogenicity was completelyabolished when a second substitution (V2(E) was included arguing that itis not an alpha-helical but an extended structure adopted by the HIV-1fusion peptide that actively destabilizes cholesterol-containing,electrically neutral membranes (Pereira et al., 1997).

The prion protein (PrP) is a glycoprotein of unknown function normallyfound at the surface of neurons and of glial cells. It is involved indiseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakobdisease in the human, where PrP is converted into an altered form(termed PrPSc). According to computer modeling calculations, the 120 to133 and 118 to 135 domains of PrP are tilted lipid-associating peptidesinserting in a oblique way into a lipid bilayer and able to interactwith liposomes to induce leakage of encapsulated calcein (Pillot et al.,1997b).

The C-terminal fragments of the Alzheimer amyloid peptide (amino acids29-40 and 29-42) have properties related to those of the fusion peptidesof viral proteins inducing fusion of liposomes in vitro. Theseproperties could mediate a direct interaction of the amyloid peptidewith cell membranes and account for part of the cytotoxicity of theamyloid peptide. In view of the epidemiologic and biochemical linkagesbetween the pathology of Alzheimer's disease and apolipoprotein E (apoE)polymorphism, examination of the potential interaction between the threecommon apoE isoforms and the C-terminal fragments of the amyloid peptideshowed that only apoE2 and apoE3, not apoE4, are potent inhibitors ofthe amyloid peptide fusogenic and aggregational properties. Theprotective effect of apoE against the formation of amyloid aggregateswas thought to be mediated by the formation of stable apoE/amyloidpeptide complexes (Pillot et al., 1997a; Lins et al., 1999).

The fusogenic properties of an amphipathic net-negative peptide (WAE11), consisting of 11 amino acid residues were strongly promoted whenthe peptide was anchored to a liposomal membrane; the fusion activity ofthe peptide appeared to be independent of pH and membrane merging andthe target membranes required a positive charge which was provided byincorporating lysine-coupled phosphatidylethanolamine (PE-K). Whereasthe coupled peptide could cause vesicle aggregation via nonspecificelectrostatic interaction with PE-K, the free peptide failed to induceaggregation of PE-K vesicles (Pecheur et al., 1997).

A number of studies suggest that stabilization of an alpha-helicalsecondary structure of the peptide after insertion in lipid bilayers inmembranes of cells or liposomes is responsible for the membrane fusionproperties of peptides; Zn²⁺, enhances the fusogenic activity ofpeptides because it stabilizes the alpha-helical structure. For example,the HEXXH domain of the salivary antimicrobial peptide, located in theC-terminal functional domain of histatin-5, a recognized zinc-bindingmotif is in a helicoidal conformation (Martin et al., 1999; Melino etal., 1999; Curtain et al., 1999).

Fusion peptides have been formulated with DNA plasmids to createpeptide-based gene delivery systems. A combination of the YKAKnWKpeptide, used to condense plasmids into 40 to 200 nm nanoparticles, withthe GLFEALLELLESLWELLLEA (SEQ ID NO:3) amphipathic peptide, which is apH-sensitive lytic agent designed to facilitate release of the plasmidfrom endosomes enhanced expression systems containing thebeta-galactosidase reporter gene (Duguid et al., 1998).

DOPE (dioleyl phosphatidyl ethanolamine) is a fusogenic lipid; elastasecleavage of N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE (SEQ ID NO:10)converted this derivative to DOPE (overal positive charge) to deliver anencapsulated fluorescent probe, calcein, into the cell cytoplasm (Pak etal., 1999). An oligodeoxynucleic sequence of 30 bases complementary to aregion of beta-endorphin mRNA elicited a concentration-dependentinhibition of beta-endorphin production in cell culture after it wasencapsulated within small unilamellar vesicles (50 nm) containingdipalmitoyl-DL-alpha-phosphatidyl-L-serine endowed with fusogenicproperties (Fresta et al., 1998).

Additional fusogenic peptides (SEQ ID NOS:4 through 9) useful in themethods of this invention are described in Table 1, below.

Fusogenic peptide Source Protein Properties Reference GLFEAIAGFIENGInfluenza virus Bongartz et WEGMIDGGGYC hemagglutinin al, 1994; HA-2YGRKKRRQRRR TAT of HIV Green and Loewenstein, 1988; the 23-residue HIV-1Was able of insert- Curtain et fusogenic N- transmembrane ing as analpha-helix al, 1999 terminal peptide glycoprotein into neutral phos-gp41 pholipid bilayers 120 to 133 and 118 prion protein tiltedlipid-associ- Pillot et to 135 domains ating peptide; inter- al, 1997bact with liposomes to induce leakage of encapsulated calcein29-42-residue Alzheimer's Endowed with Lins et al, fragment bcia-amyloidcapacities re- 1999 peptide sembling those of the tilted fragment ofviral fusion proteins nonaggregated Alzheimer's induces apoptotic Pillotet al, amyloid beta- beta-amyloid neuronal cell 1999 peptide (1-40)peptide death LCAT 56-68 lecithin forms stable beta- Peelman et helicalsegment cholesterol sheets in lipids al, 1999; acyltransferase Decout et(LCAT) al, 1999 70 residue peptide Fusion peptide Induced lipid Ghoshand (SV-t117) and N-terminal mixing of egg phos- Shai, 1999 heptadrepeat phatidylcholine/ of Sendai virus phosphatid yiglycerol (PC/PG)large unilamellar vesicles (LUVs) MSGTFGGILAGL N-terminal Was insertedinto Rodriguez- IGLL region of the S the hydrophobic Crespo et proteinof duck core of the lipid al, 1999 hepatitis B bilayer and induced Virus(DHBV) leakage of internal aqueous contents from both neutral andnegatively charged liposomes MSPSSLLGLLAG S protein of Was inserted intoRodriguez- LQVV woodchuck the hydrophobic Crespo et hepatitis B core ofthe lipid al, 1999 virus (WHV) bilayer and induced leakage of internalaqueous contents from both neutral and negatively charged liposomespeptide sequence membrane- Triggers fusion Ulrich et B18 associated seabetween lipid al, 1999 urchin sperm vesicles; a histi- protein bindindine-rich motif for binding zinc, is required for the fusogenic functionhistatin-5 Aggregates and Melino et (salivary fuses negatively al, 1999antimicrobial charged small peptide) unilamellar vesicles in thepresence of Zn²⁺ amphipathic Forms an alpha- Martin et negativelycharged helix inserted and al, 1999 peptide consisting anchored into theof 11 residues membrane (favored (WAE) at 37° C.) oriented almostparallel to the lipid acyl chains; promotes fusion of large unilamellarliposomes (LUV) A polymer of histidyl residues Midoux and polylysine(average become cationic Monsigny, 190) partially upon protonation 1999substituted with of the imidazole histidyl residues groups at pH below6.0.; disrupt endo- somal membranes GLFEALLELLESL amphipathic pep-Duguid WELLLEA tide; a pH-sensitive et al, 1998 lytic agent to facili-tate release of the plasmid from endosomes (LKKL)₄ amphiphilic fuso-Gupta and genic peptide, Kothekar, able to interact 1997 with fourmolecules of DMPC residues 53-70 (C- apolipoprotein induces fusion ofLambert et terminal helix) (apo) AII unilamellar lipid al, 1998 vesiclesand dis- places apo AI from HDL and r-HDL residues 90-111 PH-30 alpha (amembrane-fuso- Niidome et protein genic activity to al, 1997 functioningin acidic phospho- sperm-egg lipid bilayers fusion) N-terminus of NefNef protein of membrane-perturb- Macreadie human ing and fusogcnic etal, 1997 immuno- activities in arti- deficiency ficial membranes; type 1(HIV-1) causes cell killing in E. coli and yeast casein signal alpha s2-and Interact with Creuzenet et peptides beta-casein dimyristoylphos- al,1997 phatidylglycerol and -choline lipo- somes; show both lytic andfusogenic activities amino-terminal F1 polypeptide Can be used as aPartidos et sequence F1 of measles carrier system for al, 1996polypeptide virus (MV) CTL epitopes 23 hydrophobic S protein of A highdegree of Rodriguez- amino acids in the hepatitis B similarity withCrespo et amino-terminal virus (HBV) known fusogenic al, 1994 regionpeptides from other viruses. 19-27 amino acid glycoprotein Adopts anamphi- Voneche et segment gp51 of bovine philic structure al, 1992leukemia virus and plays a key role in the fusion events induced bybovine leukemia virus Ac-(Leu-Ala-Arg- basic caused a leakage of Suenagaet Leu)₃-NHCH₃ amphipathic contents from small al, 1989; peptidesunilamellar vesicles Lee et al, composed of egg 1992 yolk phosphatidyl-choline and egg yolk phosphatidic acid (3:1) amphiphilic anionic canmimic thc fuso- Murata et peptides E₅ and E₅L genic activity of al, 1991influenza hema- gglutinin(HA) 30-amino acid designed to becomes anamphi- Parente et peptide with the mimic the pathic alpha-helix al, 1988major repeat unit behavior of the as thc pH is lowered Glu-Ala-Leu-Alafusogenic to 5.0; fusion of (GALA)₇ sequences of phosphatidylcholineviral fusion small unilamellar proteins vesicles induced by GALArequires a peptide length greater than 16 amino acids pardaxinamphipathic forms voltage-gated, Lelkes and polypeptide,cation-selective Lazarovici, purified from pores; mediated the 1988 thegland aggregation of lipo- secretion of the somes composed of Red SeaMoses phosphatidylserine sole flatfish but not of phos- Pardachirusphatidylcholine marmoratus Gramicidin (linear Antibiotic; inducesMassari and hydrophobic aggregation and Colonna, polypeptide) fusion ofvesicles 1986; Tournois et al, 1990 poly(Glu-Aib-Leu- Amphiphilic struc-Kono et al, Aib) (Aib ture upon the forma- 1993 represents 2- tion ofalpha-helix; aminoisobutyric caused fusion of acid), EYPC liposomes anddipalmitoyl- phosphatidylcholine liposomes more strongly with de-creasing pH

After the micelles have been formed, they are mixed with an effectiveamount of a vesicle forming lipid to form drug containing liposomes.Useful lipids for this invention include premade neutral liposomes,lipids in powder, PEG-DSPE or hydrogenated soy phosphatidylchline(HSPC). Vesicle-forming lipids are selected to achieve a specifieddegree of fluidity or rigidity of the final complex providing the lipidcomposition of the outer layer. These can be composed of 10-60%cholesterol and the remaining amounts include bipolar phospholipids,such as the phosphatidylcholine (PC) or phosphatidylethanolamine (PE),with a hydrocarbon chain length in the range of 14-22, and saturatedwith one or more double C═C bonds. A preferred lipid for use in thepresent invention is cholesterol (10-60%), hydrogenated soyphosphatidylcholine (HSPC) at 40-90%, and the derivatizedvesicle-forming lipid PEG-DSPE at 1-7%. The liposomes provide the outerlipid bilayer surfaces that are coated with the hydrophilic polymer,PEG. The PEG chains have a molecular weight between 1,000-5,000 Dalton.Other hydrophilic polymers include hyaluronic acid,polyvinylpyrrolidone, DSPE, hydroxyethylcellulose, and polyaspartamide.PEG-DSPC and PEG-HSPC are commercially available from Syngena.

Prior to mixture with the vesicle forming lipid, the ethanol or otherorganic solvent can be removed by any method known in the art, e.g.,dialysis of the micelles through permeable membranes.

Diagnostic and Therapeutic Methods

We claim the therapy of subject, e.g., mammals such as mice, rats,simians, and human patients, with human cancers including, but notlimited to breast, prostate, colon, non-small lung, pancreatic,testicular, ovarian, cervical carcinomas, head and neck squamous cellcarcinomas. In one aspect, intravenous injection of cisplatinencapsulated into liposomes as well as by combinations of encapsulatedcisplatin with encapsulated doxorubicin, fluorodeoxyuridine, bleomycin,adriamycin, vinblastin, prednisone, vincristine, taxol or radiationtherapy, encapsulated oligonucleotides, ribozymes endowed withanticancer properties and a number of anticancer genes including but notlimited to p53/Pax5/HSV-tk genes, are claimed. Our approach consists oftwo major parts: (i) the ability to target cancer cells (ii)effectiveness of our approach to kill cancer cells.

Accordingly, this invention also provides a method for deliveringcisplatin or other therapeutic agent to a cell comprising contacting thecell with the encapsulated drugs obtainable by the methods of thisinvention. Also provided by this invention is a method for inhibitingthe growth of a tumor in a subject, comprising administering to thesubject an effective amount of the encapsulated drugs obtainable by themethods of this invention. Depending on the composition of thelipid/micelle formulation, also claimed herein are methods for targetingsolid tumors and metastases in a subject by intravenous administrationof an effective amount of the encapsulated drug and methods forpenetrating the cell membrane of a tumor in a subject by administrationof an effective amount of the encapsulated drug, wherein the micellecontains a free fusogenic peptide or a fusogenic peptide-lipidconjugate.

The methods can be practiced in vitro, ex vivo or in vitro.

In vitro practice of the method involves removal of a tumor biopsy orculturing of a cell sample containing tumor cells. The final liposomecomplex or any intermediate product arising during cisplatinencapsulation (e.g., micelles shown in FIG. 1A) are contacted with thecell culture under conditions suitable for incorporation of the drugintracellularly. The in vitro method is useful as a screen to determinethe best drug therapy for each individual patient. Inhibition of cellgrowth or proliferation indicates that the cell or tumor is suitablytreated by this therapy. Effective amount of drug for each therapyvaries with the tumor being treated and the subject being treated.Effective amounts can be empirically determined by those of skill in theart.

When delivered to an animal, the method is useful to further confirmefficacy of the drug or therapy for each tumor type. As an example ofsuitable animal models, groups of SCID mice or nude mice (Balb/c NCRnu/nu female, Simonsen, Gilroy, Calif.) may be subcutaneously inoculatedwith about 10⁵ to about 10⁹ cancer or target cells as defined herein.When the tumor is established, the liposome is administered.

As used herein, “administration, delivered or administered” is intendedto include any method which ultimately provides the drug/liposomecomplex to the tumor mass. Examples include, but are not limited to,topical application, intravenous administration, parenteraladministration or by subcutaneous injection around the tumor. Tumormeasurements to determine reduction of tumor size are made in twodimensions using venier calipers twice a week.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intravenously,intraperitoneally, subcutaneously, intrathecally, injection to thespinal cord, intramuscularly, intraarticularly, portal vein injection,or intratumorally. More preferably, the pharmaceutical compositions areadministered intravenously or intratumorally by a bolus injection. Inother methods, the pharmaceutical preparations may be contacted with thetarget tissue by direct application of the preparation to the tissue.The application may be made by topical, “open” or “closed” procedures.By “topical”, it is meant the direct application of the pharmaceuticalpreparation to a tissue exposed to the environment, such as the skin,nasopharynx, external auditory canal, eye, inhalation to the lung,genital mucosa and the like. “Open” procedures are those procedureswhich include incising the skin of a patient and directly visualizingthe underlying tissue to which the pharmaceutical preparations areapplied. This is generally accomplished by a surgical procedure, such asa thoracotomy to access the lungs, abdominal laparotomy to accessabdominal viscera, or other direct surgical approach to the targettissue. “Closed” procedures are invasive procedures in which theinternal target tissues are not directly visualized, but accessed viainserting instruments through small wounds in the skin. For example, thepreparations may be administered to the peritoneum by needle lavage.Likewise, the pharmaceutical preparations may be administered to themeninges or spinal cord by infusion during a lumbar puncture followed byappropriate positioning of the patient as commonly practiced for spinalanesthesia or metrazamide imaging of the spinal cord. Alternatively, thepreparations may be administered through endoscopic devices.

Administration in vivo can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with thecomposition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents can be empirically determined bythose of skill in the art.

The agents and compositions of the present invention can be used in themanufacture of medicaments and for the treatment of humans and otheranimals by administration in accordance with conventional procedures,such as an active ingredient in pharmaceutical compositions.

Ideally, the drug/lipid formulation should be administered to achievepeak concentrations of the active compound at sites of disease. This maybe achieved, for example, by the intravenous injection of the drug/lipidformula. Desirable blood levels of the drug may be maintained by acontinuous infusion to provide a therapeutic amount of the activeingredient within disease tissue. The use of operative combinations iscontemplated to provide therapeutic combinations requiring a lower totaldosage of each component drugs than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

While it is possible for the drug/lipid formula to be administeredalone, it is preferable to present it as a pharmaceutical formulationcomprising at least one active ingredient, as defined above, togetherwith one or more pharmaceutically acceptable carriers therefor andoptionally other therapeutic agents. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient.

Designing the third generation of vehicles for the delivery ofanticancer drugs and genes to solid tumors at as described herein wasthe result of five major improvements over existing technologies:

1.) Encapsulation of antineoplastic drugs into sterically stabilizedliposomes has reduced manifold their toxicity. This is anticipated tobring to an end the nightmare of cancer patients subject tochemotherapy. Most antineoplastic drugs under current use have severeside effects such as hair loss, vomiting, weight loss and causeinfarction as well as damage to kidneys, brain, liver and all othervital tissues. The antineoplastic drugs described herein are hiddeninside the lumen of the lipid bilayer, are not visible to most tissuesand concentrate into their tumor targets, not in every tissue in thebody. Upon their uptake by the solid tumor they exert a specificcytotoxic effect to cancer cells without damaging normal cells.

2.) Targeting of solid tumors and their metastases all over the body.Over 95% of cancer patients succumb from complications connected tometastases, not from the primary tumor. Our gene and drug deliverysystem hag been designed to evade the immune system after intravenousadministration of the gene and drug bullet reaching not only the primarytumor but also every metastasis in the animal and human body regardlessof the size of the tumor. It is based on the long circulation time ofour drug and gene carrying vehicles and their extravasation through thevascular endothelium of tumors because of its imperfections andleakiness at its initial stage of formation (neoangiogenesis in growingtumors) as well as because of differences in hydrostatic pressurebetween the growing solid tumor and normal body tissues. The liposomesof this invention have a different composition between their inner andouter lipid layers permitting efficient encapsulation and tumortargeting.

3.) Uptake of the liposome bullet by the cancer cell. The liposomebullets are able to promote fusion with the cell membrane. Similar“stealth” bullets developed elsewhere are unable to cross the membranebarrier of the cancer cell.

4.) Reaching nearly 100% liposome encapsulation efficiency foranticancer drugs, oligonucleotides and genes is a major advancement.This means minimal loss and cost effective use of drugs and genes. Italso translated into simpler steps in manufacturing the anticancerbullet.

5.) The unique technology described herein can identify regulatory DNAsequences that sustain expression of the genes in the anticancer bulletfor months rather than days. This translates into fewer treatments andless suffering of the cancer patient. It can also exert a strongtherapeutic effect because of higher levels of expression of theanticancer gene; the same gene placed under control of weak regulatoryDNA will be ineffective.

The following examples are intended to illustrate, and not limit theinvention.

EXAMPLES

Preparation of Micelles and Lipid-Encapsulated Cisplatin

One formula for encapsulation includes the steps of: (A) mixingcisplatin (in powder or other form) with DPPG (dipalmitoyl phosphatidylglycerol) or other negatively-charged lipid molecules at a 1:1 to 1:2molar ratio in at least a 30% ethanol, 0.1 M Tris HCl, pH 7.5 to achieveabout 5 mg/ml final cisplatin concentration. Variations in the molarratio between cisplatin and DPPG are also of therapeutic value targetingdifferent tissues. (B) Heating at 50° C. During steps A and B theinitial powder suspension, which tends to give a precipitate of theyellow cisplatin powder, is converted into a gel (colloidal) form;during steps A and B there is conversion of cisplatin to its aqua form(by hydrolysis of the chloride atoms and their replacement by watermolecules bound to the platin) which is positively-charged and is theactive form of cisplatin endowed with the antineoplastic activity; theaqua cisplatin is simultaneously complexed with the negatively-chargedlipid into micelles in 30% ethanol. This cisplatin-DPPG electrostaticcomplex has already improved properties over free cisplatin in tumoreradication. (C) The properties of the complex (and of the finalformulation after step D, see below) in passing through the tumor cellmembrane after reaching its target are improved by addition of peptidesand other molecules that give to the complex this property. (D) Thecisplatin-DPPG micelle complex is converted into liposomes encapsulatingthe cisplatin-DPPG monolayer (FIG. 1 top) or to other type of complexesby direct addition of premade liposomes followed by dialysis againstsaline and extrusion through membranes to downsize these to 100-160 nmin diameter (FIG. 1 bottom). It is the lipid composition of addedliposomes that determines the composition of the outer surface of ourfinal cisplatin formulation.

Variations in step (A) permit encapsulation of doxorubicin and otherpositively charged antineoplastic compounds. Addition of positivelycharged groups to neutral or negatively-charged compounds allows theirencapsulation similarly into liposomes.

Therapeutic Application

Ninety (90) day-release estrogen pellets were implanted subcutaneouslyinto SCID female mice. The mice were subcutaneously injected at mammaryfat pad with 7.5 million MCF-7 (a human breast carcinoma available fromthe ATCC) cells in 0.1 ml PBS. After establishment of tumors, the micewere injected intravenously at tail vein with 0.1 ml of cisplatinliposomes. Results are shown in FIGS. 2 to 4.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

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10 1 24 PRT Influenza virus hemagglutinin HA-2 1 Gly Leu Phe Glu Ala IleAla Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly GlyGly Tyr Cys 20 2 11 PRT Human Immunodeficiency Virus 2 Tyr Gly Arg LysLys Arg Arg Gln Arg Arg Arg 1 5 10 3 20 PRT Artificial Sequence A fusionpeptide formulated with DNA plasmids to create peptide-based genedelivery systems. 3 Gly Leu Phe Glu Ala Leu Leu Glu Leu Leu Glu Ser LeuTrp Glu Leu 1 5 10 15 Leu Leu Glu Ala 20 4 16 PRT Duck Hepatitis B Virus4 Met Ser Gly Thr Phe Gly Gly Ile Leu Ala Gly Leu Ile Gly Leu Leu 1 5 1015 5 16 PRT Woodchuck hepatitis B virus 5 Met Ser Pro Ser Ser Leu LeuGly Leu Leu Ala Gly Leu Gln Val Val 1 5 10 15 6 20 PRT ArtificialSequence An amphipathic peptide. 6 Gly Leu Phe Glu Ala Leu Leu Glu LeuLeu Glu Ser Leu Trp Glu Leu 1 5 10 15 Leu Leu Glu Ala 20 7 16 PRTArtificial Sequence An amphiphilic fusogenic peptide. 7 Leu Lys Lys LeuLeu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu 1 5 10 15 8 12 PRTArtificial Sequence Basic amphipathic peptide. 8 Leu Ala Arg Leu Leu AlaArg Leu Leu Ala Arg Leu 1 5 10 9 28 PRT Artificial Sequence 30 aminoacid peptide with the major repeat sequence of this sequence, designedto mimic the behavior of the fusogenic sequences of viral fusionproteins. 9 Gly Ala Leu Ala Gly Ala Leu Ala Gly Ala Leu Ala Gly Ala LeuAla 1 5 10 15 Gly Ala Leu Ala Gly Ala Leu Ala Gly Ala Leu Ala 20 25 10 4PRT Artificial Sequence Fusogenic peptide. 10 Ala Ala Pro Val 1

What is claimed is:
 1. A method for producing cisplatin micelles,comprising: a) combining a suitable buffer solution, cisplatin with aneffective amount of at least a 30% ethanol solution to form acisplatin/ethanol solution; and b) combining the solution with anegatively charged phosphatidyl glycerol lipid derivative wherein themolar ratio between cisplatin and the lipid derivative is 1:1 to 1:2,thereby producing a cisplatin mixture in its aqua form in micelles.
 2. Amethod of producing cisplatin micelles, comprising: a) combining asuitable buffer solution, cisplatin with an effective amount of at least30% ethanol solution to form a cisplatin/ethanol solution; and b)combining the cisplatin/ethanol solution with a negatively chargedphosphatidyl glycerol lipid derivative wherein the molar ratio betweencisplatin and the lipid derivative is 1:1 to 1:2, thereby producing acisplatin mixture in its aqua form in micelles.
 3. The method of claim 1or 2, wherein the phosphatidyl glycerol lipid derivative is selectedfrom the group consisting of dipalmitoyl phosphatidyl glycerol (DPPG),dimyristoyl phosphatidyl glycerol (DMPG), dicaproyl phosphatidylglycerol (DCPG), distearoyl phosphatidyl glycerol (DSPG) and dioleylphosphatidyl glycerol (DOPG).
 4. The method of claim 1 or 2, wherein themolar ratio is 1:1.
 5. The method of claim 1 or 2, further comprisingcombining an effective amount of a free fusogenic peptide, a fusogenicpeptide-lipid conjugate or a fusogenic peptide—PEG-HSPC conjugate to themixture of step a) where the fusogenic peptide is derivatized with astretch of 1-6 negatively-charged amino acids at the N or C-terminus andthus, able to bind electrostatically to the cisplatin mixture in itsaqua form.
 6. The method of claim 5, wherein the free fusogenic peptideor fusogenic peptide lipid conjugate comprises DOPE or DOPE/cationiclipid.
 7. The cisplatin micelle obtained by the method of claim
 5. 8.The cisplatin micelle obtained by the method of claim 1 or
 2. 9. Amethod for penetrating the cell membrane of a tumor cell in a subjectcomprising administering an effective amount of the cisplatin micelleobtainable by the method of claim
 8. 10. A method for encapsulatingcisplatin micelles, comprising mixing an effective amount of avesicle-fonming lipid with the cisplatin micelles of claim 1 or
 2. 11.The encapsulated cisplatin lipid micelle obtainable by the method ofclaim
 10. 12. A method for obtaining a cisplatin/lipid complex capableof evading macrophages and cells of the immune system when administeredto a subject, the method comprising mixing an effective amount of thecisplatin micelles of claim 11 with an effective amount of lipidselected from the group consisting of PEG-DSPE, PEG-DSPC and hyaluronicacid—DSPE.
 13. An encapsulated cisplatin/lipid complex obtainable by themethod of claim
 12. 14. A method for delivering cisplatin to a cellcomprising contacting the cell with the encapsulated cisplatin/lipidcomplex of claim
 13. 15. A method for inhibiting the growth of a tumorin a subject, comprising administering to the subject an effectiveamount of the encapsulated cisplatin/lipid complex of claim
 13. 16. Acomposition comprising the encapsulated cisplatin micelle of claim 11and encapsulated oligonucleotides, ribozymes, triplex, PNA.
 17. Acomposition comprising the encapsulated cisplatin micelle of claim 11and a drug selected from the group consisting of doxorubicin,fluorodeoxyuridine, bleomycin, adriamycin, vinblastin, prednisone,vincristine, taxol.
 18. The method of claim 10, wherein the lipid isselected from the group consisting of pre-made neutral liposomescomprising 10%-60% cholesterol, 40-90% hydrogenated soyphosphatidylcholine (HSPC), 1-7% polyethyleucglycol (PEG)-HSPC andPEG-DSPE.
 19. An encapsulated cisplatin lipid micelle obtainable by themethod of claim
 18. 20. A method for delivering cisplatin to a cellcomprising contacting the cell with the encapsulated cisplatin lipidmicelle of claim
 19. 21. A method for inhibiting the growth of a tumorin a subject, comprising administering to the subject an effectiveamount of the encapsulated cisplatin lipid micelle of claim
 19. 22. Amethod for targeting solid tumors and metastases in a subject comprisingintravenous administration of an effective amount of the encapsulatedcisplatin micelle of claim 19 or the cisplatin/lipid complex of claim13.
 23. The method of claim 10, wherein the lipid comprises 10-60%cholesterol.
 24. The method of claim 10, wherein the vesicle-forminglipid is in solution or powder form.
 25. The method of claim 1 or 2,further comprising removal of the ethanol from the cisplatin micelles.26. The method of claim 25, wherein removal of the ethanol is bydialysis of the cisplatin micelles through permeable membranes to removethe ethanol.